Not Applicable.
The present invention relates to a phototriggerable composition and method for use in crosslinking protein. More specifically, the present invention relates to a tethered diazopyruvate composition for use in crosslinking collagen, whereby the composition results in sutureless wound closure, for example, of a tendon or cornea.
There is a desire to replace sutures in ocular surgery with a light-activated collagen bonding agent. Elimination of sutures by use of such a bonding agent would be of benefit primarily in corneal procedures. The number of penetrating keratoplasties performed in the United States is steadily increasing, with more than 30,000 being performed in 1994. Overall, penetrating keratoplasty is a highly successful procedure with reported graft survival rates of 79% to 90% after 3-5 years. Although thousands of successful corneal transplants are performed each year, there are significant drawbacks to the present surgical procedure. The major problem is that opposed stroma of the donor and recipient corneas do not fully and uniformly rejoin. Rather, sporadic fibrous scar tissue is formed at different locations along the wound by activated keratocytes as part of the corneal wound healing process. The fibrous tissue crosses from one side of the incision to the other, but the original lamellar structure is never reestablished. Furthermore, the degree of fibrous interconnection can vary from one area of the incision to another so that when the sutures are removed, there may be localized wound gaping and consequent astigmatism. The lack of fully rejoined collagen results in a weakened corneal structure, and consequently, blows to the cornea can result in wound dehiscence and severe ocular damage. Although wound dehiscence is not the major cause of graft failure, it poses a significant problem. For example, it has been reported that about 4% of wound failures are due to wound dehiscence and leakage.
A second major problem with the present surgical method concerns the sutures themselves. First, the suture material can initiate an inflammatory response with resulting pain, irritation, and invitation of blood vessels. Second, suturing makes a hole in the cornea that allows entry of infectious agents that are normally excluded by the epithelium. Third, sutures do not apply even pressure along the circumference of the wound. Areas of the wound that are not firmly held together are susceptible to leakage of aqueous humor and epithelial downgrowth. Together, the problems of inflammation, irritation, wound gape, leakage and epithelial downgrowth pose significant clinical problems in postsurgical management of corneal transplant and are frequent causes for transplant failure. It is known that the most common complications after graft placement are related to the sutures. In fact, it has been observed that 25% of patients experience suture complications including exposed sutures, stitch abscess, breakage and loosening, and wound dehiscence.
Sutures are also a frequent cause of post-operative visits to emergency rooms. For example, it has been observed that 14.4% of patients with corneal or conjunctival monofilament nylon sutures make two or more extra visits to the emergency room due to ocular irritation caused by loosened or broken sutures. For these reasons, it is desired to have a composition or method, which can be used in place of known surgical methods. Such method or composition will preferably eliminate the need for sutures.
It is further desired to be able to repair other types of collagenous tissue. Surgery on other bodily collagenous tissues may benefit from development of a phototriggerable collagen crosslinker. For example, tendon repair would seem an ideal candidate because, like the cornea, tendon is primarily Type I collagen, has little vascularization, and normally contains few active cells. Surgical repair of tendons occurs as a treatment for injury or as a corrective measure for patients with neurological deficits with accompanying abnormal tone, pulling an extremity into an undesirable position. The most commonly performed surgical procedures for stroke patients are: (1) tendon lengthening or release, (2) soft-tissue release, and (3) tendon transfers. Furthermore, tendons are the most prevalent site of surgery for the correction of overuse or chronic repetitive use problems (32% of all surgery relating to overuse is for tendons).
Tendon healing is a slow process, requiring at least six weeks, that may lead to extended hospitalization times with prolonged inactivity resulting in long-lasting morbidity. Human studies have shown that six months following surgery, the Achilles tendon has not recovered concentric or eccentric plantar flexion muscle strength compared to non-injured tendons. Following ligament-tendon autografts, low mechanical strength has been reported in the tendons of various animal models as long as one to three years after surgery. In addition, it has been found, using a rabbit, Achilles tendon model, that two weeks following reapproximation with sutures, tendons have less than half the strength of intact tendons and the decreased strength remains for at least 6 months. Clinically, prolonged healing of sutured tendons requires long periods of immobilization, predisposing patients to muscle atrophy, joint cartilage ulceration, tendo-cutaneous adhesion, and rerupture. What is desired is a composition that improves the tendon healing because it would provide immediate collagen-to-collagen bonds that otherwise are only re-established through the long-term wound healing mechanism when sutures alone are used for repair. It is especially desired to have a composition that eliminates the need for sutures.
For phototriggerable, collagen-crosslinking compounds to be useful in surgical wound closure, light of the appropriate wavelength must penetrate the tissue deeply enough to activate all of the material while not damaging the tissue. Previously developed phototriggerable compounds are activated in the 250 nanometer (nm) to 350 nm range, a spectral range potentially damaging to the eye. Therefore, an important design criterion is to select chromophores that can be activated at wavelengths that produce sufficient penetration with minimal phototoxicity. For the eye, this wavelength range is approximately 330 nm to 400 nm since absorption by cornea is quite low, and phototoxic effects are minimal. Thus, by proper selection of the chromophore, it is possible to design and synthesize phototriggerable compounds that are activated at wavelengths that will penetrate collagenous tissues deeply, yet cause minimal phototoxic effects.
The intact cornea is approximately 78% water by weight, hence phototriggerable crosslinkers must readily penetrate the aqueous environment of the tissue in order to form bonds with collagen. Tendon, sclera, and conjunctiva also have relatively high water contents. Consequently, an important design consideration for phototriggerable crosslinkers is to make them hydrophilic.
Previous experience with 1,8-naphthalimide dye suggests that this compound is extremely quantum inefficient; that is, the number of chemical bonds formed per incident photon is quite low. This, in turn, necessitates extremely high total energies to form tissue bonds, which is problematic for realistic surgical application. Thus, a suitable phototriggerable crosslinker, should have a relatively high quantum efficiency.
The present invention relates to a photoactivated composition for crosslinking protein wherein the composition is a diazopyruvate of the general formula RHNCOCOCHN2 where R is selected preferably from the group consisting of oligopeptides and polyethylene glycol. Generally, the composition is described as a tethered photoactivated compound having two diazopyruvate molecules. The preferred composition to be photoactivated is an N,Nxe2x80x2-bis(3-diazopyruvoyl)-2,2xe2x80x2-(ethylenedioxy)bis(ethylamine) that is in a solution and can be applied to the tissue. The crosslinked protein is, more specifically, a collagen composition. Activation of the photoactive composition occurs at a wavelength that is non-damaging to the surrounding tissue. Suitable wavelengths range between 330 nm and 400 nm. Preferably, the composition is hydrophilic and non-toxic. The composition used to tether the photoactive compound is selected from the group consisting of polyethylene glycol and oligopeptides.
The present invention also relates to a method for crosslinking proteins. The method includes: forming a hydrophilic, non-toxic photoactive composition; applying the composition to collagenous tissue; and, irradiating such composition at a wavelength of between 300 nm and 400 nm, whereby the tissue wound is repaired. Irradiation forms a reactive intermediate composition that forms covalent bonds with the collagen. The intermediate is a ketoketene and will bond with the free amine of an amino acid such as lysine. The resultant crosslinked composition is typically a bis-1,n-malonylamide.